The techineue of cloning human tumor stem cells in soft agar appears to be a promising tool for testing the sensitivity of tumor cells to different chemotherapeutic drugs. The data available at present, though encouraging, does not include solid tumors, but mainly pertains to hemopoietic tumors and malignant effusions. Clonogenicity of tumor cells from solid neoplasms needs to be improved before this technique can be routinely used for testing the efficacy of chemotherapeutic agents. This proposal is conceived to explore methods of improving clonogenicity of human colo-rectal tumor stem cells. We have developed a method to separate viable from non-viable cells in disaggregated solid tumors by Ficoll-Hypaque centrifugation. We intend to examine various viable cell plating concentrations in the assay, and to study the interactions of viable and non-viable cells in the platings. Various exogenous factors that have been reported to improve growth will be examined, e.g., macrophages, epidermal growth factor, prostaglandins, and platelets. Another approach which we propose to utilize is the combination of radiation and drugs that may improve the specificity and efficacy of anti-tumor chemotherapy. This is based on the observation that irradiation increases the proliferative activity of a tumor, in stimulating many dormant cells out of the Go state, leading to enhanced sensitivity to stage specific drugs and anti-metabolites. Finally, the Courtenay procedure for culturing tumor cells developed in England, which utilizes red blood cells from the August rat as a feeder factor, will be compared with the Dalmon assay on the same batch of tumor stem cells. A number of cell lines developed from human colo-rectal tumors are now available. Colo-rectal tumor cells produce carcinoembryonic antigen (CEA), a convenient biochemical marker of differentiation, which can be exploited in these studies for identification purposes. These were persuasive reasons to focus on colo-rectal tumors for studies on human tumor cloning.